Method for land improvement and microorganisms therefor

ABSTRACT

The invention relates to methods for land improvement (soil reclamation), preferably for soil remediation comprising the steps of (i) placing a material useful for improving soil, and an explosive in the soil, and (ii) mixing the materials useful for reclaiming the soil and the polluted soil by explosion. The invention also relates to methods methods for providing microorganisms useful for decomposing hydrophobic pollutants, mineral oil components and derivatives by selection and isolation from soil, the microorganisms, their uses and kits for land improvement and soil remediation. The invention is useful in particular for remediation of soil polluted with oil components and derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Stage filed under 35 U.S.C. § 371 which claimsthe benefit of priority to International Patent Application No.PCT/HU02/00103 filed Oct. 8, 2002, which claims the benefit of priorityto Hungarian Patent Application No. P0203394 filed Oct. 7, 2002 andHungarian Patent Application No. P0104154 filed Oct. 8, 2001, thedisclosures of which are incorporated herein in full by reference.

DESCRIPTION

The invention relates to land improvement by mixing the soil andmaterials useful for reclaiming it using explosion. Preferably theinvention relates to methods for reducing the extent of soil pollutionby using microorganisms selected for this purpose. The invention alsorelates to methods for providing the microorganisms in an isolated form,the microorganisms themselves, their uses and kits for land improvementand soil remediation.

The method for soil remediation preferably comprises placing in thepolluted soil or close to it, below the surface of the soil,microorganisms useful for decomposing or inactivating at least one kindof material, said microorganisms being effective both in aerobic andanaerobic conditions, and an explosive and mixing the polluted soil, themicroorganisms and, optionally, additives for improving livingconditions of microorganisms by explosion, and allowing themicroorganisms to act in the soil.

The polluted soil in most cases is cleansed with living organisms,usually microorganisms useful for that purpose (1, 2).

Effective bioremediation with microorganisms can be achieved ifmicroorganisms (11) resistant to the certain pollutant and capable ofdecomposing it, are used in their required growth conditions(temperature, moisture, oxygen concentration, nutrition etc.). Thus, itis important to add additives which influence the microorganisms'function and the effectiveness of bioremediation to their advantage (forexample micro and macro elements, carbon, nitrogen, phosphorous,sulphur, Mn, etc.) (11).

It is important to disperse or spread the essential additives and themicroorganisms used in bioremediation in the polluted medium. Severalsolutions to this problem exist up to date.

In case of soils, traditionally the additives and microorganismsadministered to the surface are filtered (using gravity), injected ormixed into the soil. (5, 6) These technologies provide inadequatemixing, or the required distribution of materials in the polluted soildevelop only very slowly. In addition the process is hardlycontrollable.

In another method used in practice regularly the acquired polluted soilis mixed with materials promoting its decomposition, then this mixtureis brought back to the field (prismatic technology). This method isquite expensive, especially in case of larger areas or in case ofcleansing low-lying pollutions.

The invention is based on the unexpected finding that theabove-mentioned problems can be economically and effectively solved bymixing the soil and microorganisms chosen for the given purpose (and,preferably, further additives) applying explosion. The novel method wasnamed bio-explosion.

According to the art there are microorganisms known for decomposingvarious pollutants, mineral oil components or derivatives and methodsfor selecting these, wherein the principle of said methods is thatmicroorganisms are supplied with the material to be decomposed as theonly carbon source. To the best of our knowledge tenside producingability of the microorganisms was not examined.

Such microorganism are e.g. those which are commercialized by OilCleaning Bio-Products Ltd. P.O.Box 46, Royston, Hertfordshire SG8 9PDU.K., e.g. Hegrem and Hegboost products (e.g. the enclosed productdescriptions).

However, a need for advantageous selection methods, which lead toeffective microorganisms with a high reliability and which include clearselection criteria, still exists.

DEFINIIONS

The term “soil” includes the whole depth of the soil from the surfacelayers (A-level or humus level) to the deepest layer in contact with theparent material or the impermeable layer (e.g. geologic level orD-level).

The phrase that holes are arranged “essentially alternately” relates toan arrangement of holes according to which no holes with identicalfilling can be found in the vicinity of each other in a large number,preferably at most 5 or more preferably at most 3 holes with anidentical filling can be found in the vicinity of each other.Preferably, in the major part of the area with holes the holes arealternating according to a simple mathematical rule, e.g. no holes withan identical filling can be found in the vicinity of each other.

The term “essentially regular distances” is used herein in connectionwith an arrangement of holes according to which on a given part of thearea comprising holes the distance of holes from each other isessentially identical i.e. the distances vary in at most 50%, 40%, 30%,20%, highly preferably in at most 10 or 5%, at least in one direction,preferably in all directions in the plane.

A “mineral oil component” is meant herein as any component, fraction orany mixture thereof of the raw mineral oil.

A “mineral oil derivative” is meant herein as any artificiallypreparable derivative of the mineral oil or any component thereof, or aderivative produced in a non-geological process.

“Tenside” means any surfactant.

The term “microorganism” is meant herein as living organism, either ofmono or multicellular structure or without and cellular structure,preferably monocellular organisms, which belong to the scope ofmicrobiology. “Microorganisms” are preferably algae, in particular bluealgae; bacteria and fungi.

A “microorganism strain” is a pure culture of microorganisms startedfrom a single cell, preferably a culture of a given species maintainedor maintainable by regular subculturing.

BRIEF DESCRIPTION OF THE INVENTION

Thus, in an aspect the invention relates to a process for improvingquality of soil said process comprising the steps of

-   -   (i) placing    -   a material useful for improving soil, and    -   an explosive    -   in the soil, and    -   (ii) mixing the materials useful for improving (reclaiming) soil        and the polluted soil by explosion.

Preferably, by the process of the invention the degree of the pollutionis decreased by

-   -   (i) placing in the polluted soil or close to it, below the        surface of the soil    -   microorganisms useful for decomposing or inactivating at least        one kind of material, which are, if desired, resistant to said        material,    -   an explosive and    -   preferably    -   additive materials improving living conditions of microorganisms        and/or facilitating their propagation and/or soil-improving        function,    -   (ii) mixing the polluted soil and the placed materials and        microorganisms by explosion,    -   (iii) allowing the microorganisms to act in the soil.

Preferably the microorganisms, explosive and optionally furthermaterials are placed in holes bored in soil, preferably at a distance of0.5 to 5 m, preferably 1.5 to 2 m from each other and preferably themicroorganisms and the explosive are placed in separate holes. Holescontaining explosive and holes not containing it can be located e.g.essentially alternately and preferably at essentially regular distances,more preferably in rows, even more preferably arranged according to ageometric network.

Preferably, after the explosion additives improving living conditions ofthe microorganisms and/or facilitating decomposition are introduced intothe soil, for instance by aeration, infiltration or injection. Aspreferred additives one or more of the following are applied:

-   -   compounds promoting anoxic respiration, preferably redox systems        and/or electron acceptors and/or hydrogen acceptors, more        preferably compounds of metals with more than one oxidation        state and/or nitrite-, nitrate-, chlorite-, chlorate-,        perchlorate-, phosphate-, pyrophosphate-, sulphite-, sulphate-,        pyrosulphate-ions or their salts.    -   metal ions, trace elements enhancing enzyme activity, in        particular Fe-, Cu-, Ni-, Co-, Mn-, Mg-, Zn-, or Ca-ions,    -   carbon sources, preferably glucose, sacharose, molasses,        glycerol, acetate, xantane,    -   nitrogen sources, preferably peptone, nitrite, nitrate, ammonium        ions or their salts,    -   phosphorous sources, preferably phosphate, pyrophosphate ions or        their salts,    -   sulphur sources: sulphate, pyrosulphate ions or their salts,    -   tensides and/or surfactants, preferably Tween 20, Tween 40,        Tween 60, Tween 80, nonite, DMSO,    -   compounds promoting adhesion to surface which are preferably        biologically degradable, xantane.

The strength of explosion is preferably set to a value which results inthe damage of at most a small part of microorganism, more preferably inno damage and no uncovering of the microorganisms.

According to a highly preferred embodiment the pollutant is a mineraloil component or derivative.

In a further aspect the invention relates to a process for preparing amicroorganism in an isolated form, said microorganism being useful fordecomposing a hydrophobic pollutant, mineral oil component or derivativeand capable of exerting its decomposing activity at the boundary of thehydrophobic phase comprising the said hydrophobic pollutant, mineral oilcomponent or derivative and a hydrophilic phase, the process comprisingthe steps of

-   -   i) applying a film comprising the mineral oil component or        derivative to a minimal medium lacking carbon source,    -   ii) inoculating this medium with a sample comprising a mixture        of microorganisms said sample being obtained from an oil        pollution, and incubating the medium after inoculation at least        until detectable microorganism colonies are formed,    -   if the formation of colonies does not occur within an        arbitrarily defined time period step i) and present step ii) are        repeated,    -   iii) decomposing activities of the microorganism from the        colonies formed are tested at the surround of the colonies and    -   iv) tenside producing abilities of the decomposing        microorganisms obtained from the colonies are checked and a        tenside producing microorganism is selected.

Preferably, the microorganism is a facultative anaerobe which isobtained by using minimal medium comprising materials facilitatinganoxic respiration, preferably electron acceptors and/or oxygensources—in particular one or more of the following: Ti-compounds,Mn-compounds, nitrite, nitrate, phosphate, pyrophosphate ions or theirsalts, and preferably the incubation is carried out at least partlyunder anaerobic conditions.

In a preferred embodiment decomposing activity is assessed by assayingthe pollutant concentration of samples taken from the closesurrounding/immediate vicinity of the colonies and/or on the basis ofthe diameter of the decomposed area.

As a decomposing activity e.g. paraffin decomposing activity can beassayed or an enzyme activity for decomposing typical mineral oilpollutions, preferably by sampling, solvent extraction then by gaschromatography. Tenside producing ability of the microorganisms from thecolonies obtained can be studied by e.g. a hydrophobic-hydrophilic droptest.

In a further aspect, the invention relates to a microorganism useful fordecomposing a mineral oil component or derivative, capable of exertingits decomposing activity at the boundary of the hydrophobic phasecomprising the said hydrophobic pollutant, mineral oil component orderivative and a hydrophilic phase, said microorganism producing atleast one enzyme capable of decomposing the mineral oil component orderivative, and at least one tenside.

Preferably the microorganism is a strain belonging to the Bacillussubtilis species, the Bacillus cereus species, the Pseudomonas genus orthe Xanthomonas genus and is, preferably, a facultative anaerobe.

Highly preferably the oil-pollutant decomposing activity of themicroorganism, detected by culturing on a polluted medium on any of thefollowing oil-pollutants: hydrophobic deposit, asphaltene, maltene, 5%asphaltene plus oil, is at least 1.2 times, preferably 1.5 or 2 times,highly preferably 3 times larger, as an average, than that of the Hegremor Hegboost microorganism.

The invention relates to microorganisms obtainable by the process of theinvention, preferably any of the following strains deposited on Apr. 17,2002 at the NCAIM: NCAIM (P) B 1304, NCAIM (P) B 1305, NCAIM (P) B 1306,NCAIM (P) B 1307, NCAIM (P) B 1308, or any strain derived therefrom.

The microorganism may be genetically modified, preferably may carry,incorporated into their genome, a DNA fragment of a known sequence.

In a further aspect the invention relates to the use of a microorganismof the invention for decomposing a soil pollution caused by a mineraloil component or derivative.

In a further aspect the invention relates to a kit for soil remediationor land improvement (soil reclamation) comprising an information carrierwith users instructions which comprise instructions for carrying out anyof the steps of any of the processes of claims 1 to 8 and said kitfurther comprising at least one kind of material applicable in any ofthe processes of claims 1 to 8.

The kit comprises preferably a microorganism according to the invention,and more preferably further comprises on or more of the following group:explosives, aids to blasting, additives to ensure living conditions orhelping microorganisms or increasing the effect thereof. Highlypreferably the kit comprises one or more kind of the above-definedadditives.

BRIEF DESCRIPTION OF THE FIGURES

In FIG. 1, colonies of isolated bacteria can be seen streaked(inoculated) on a thin film of pollutant in three Petri-dishes. It canbe observed that the pollutants are decomposed or converted surroundingthe colonies. This can be seen as clearing or discoloration around thecolonies. Whether we want to characterize the activity of decomposition,we can measure the width (diameter) of the cleared up (discolored) band.

In FIG. 2 the ability of the acquired microorganism strains (phyla) toproduce tensides is examined. With the hydrophilic—hydrophobic drop testone can observe the difference between spreading and non-wetting drops.

FIG. 3 shows the effect of several microorganism strains—described usingchromatography—on the hydrocarbon content of the paraffin sample (for V.see FIG. 3 a, for II. see FIG. 3 b) after one week of incubation. In thebar chart the ratio (expressed in percentage) the area below the curvecharacteristic of the undecomposed sample can be seen in the ratio ofthe area below the curve of the whole undecomposed mass. The marks onthe horizontal axis mean the following microorganism strains. Ref IHegrem* Ref II Hegboost* A MOL-2 NCAIM (P) B 1304 B MOL-32 NCAIM (P) B1305 C MOL-51 NCAIM (P) B 1306 D MOL-66 NCAIM (P) B 1307 E MOL-107 NCAIM(P) B 1308 F MOL-113 A Pseudomonas sp. strain isolated by the inventors*commercialized by Oil Cleaning Bio-Products Ltd. P.O.Box 46, Royston,Hertfordshire SG8 9PD U.K., see also the product descriptions and thehome page: www.ocbp.co.uk.

In FIGS. 4.a and 4.b, a possible concrete arrangement of the holes boredin the soil is shown. Microorganisms can be placed in the same or indifferent holes.

In FIG. 5.a we can see the decrease in pollutants in the soil expressedin percentage, as a function of time. In FIG. 5.b the steps of inlandexperiments can be seen.

DETAILED DESCRIPTION OF THE INVENTION

Below by non-limiting examples, some embodiments of the invention aredescribed, for example:

-   -   (i) conditions of the blast, materials and measures        advantageously affecting living conditions of the microorganisms        in the blasted soil and    -   (ii) selection method used for selecting microorganisms useful        for soil bioremediation and the properties of preferred        microorganisms.    -   (i) The soil treated by bio-explosion is highly scarified        (mellowed) due to the intervention. The strength of the blast        can be determined depending on the structure of the soil, on the        concentration of the pollutant and its location in depth        calculated from the surface and on the area of the pollution. It        is expedient to aim at fully mixing the microorganisms and the        materials ensuring or enhancing their vital functions (the        “additives”) with the polluted soil, while avoiding that the        components get to the surface of the soil. To that purpose a        blast of calculated strength and, by all means, a moderate        strength is needed.

In most cases it is expected to be preferred if microorganism andadditives are evenly spread and also the strength of the explosion ispossibly evenly distributed. Of course, if the quality of the soil orthe distribution of the pollutant in not homogenous, other aspects areto be considered.

Thus, in the process of the invention the microorganism, the explosiveand optionally further materials are placed in holes bored in the soilwhich are found at a distance of 0.5 to 5 m, preferably 1 to 3 m, morepreferably 1.5 to 2 m from each other. By the distance of the holes andthe quantity of the microorganisms the effectness of the bioremediationcan be controlled as required by the pollutant.

Microorganisms, explosive and, if desired, additives can be placed inthe same hole (in such cases expediently the explosive is placed below)provided that at the site of the explosion a sufficient quantity ofmicroorganisms survive so that the desired remediation effect could beachieved. (In certain cases a protective layer can be applied to protectmicroorganisms.) More preferably the explosive and the microorganism(s)[if desired, together with the additive(s)] can be placed in separateholes. Highly preferably the explosive, the microorganisms and theadditives are each placed in separate holes.

The depth of the holes, the geometry of the microorganisms, additivesand the explosive in them and the strength of the explosion is set sothat a damage, at least a damage larger than necessary, and getting thematerials to the surface could be avoided (see Example 5). A preferredexplosion is in most cases relatively mild.

In a preferred embodiment the holes are placed at essentially regulardistances, preferably according to an essentially regular geometry, e.g.in rows and/or in columns. Highly preferably the holes comprising andnot comprising explosive are arranged essentially alternately. Forexample, if holes with two types of filling (e.g. microorganism andexplosive) are applied a checkerboard pattern, if three types (e.g.microorganism, explosive and additive), a triangulated pattern ispreferred.

Blasting can be carried out even in the groundwater, below thegroundwater surface if the blasting material is rendered waterproof, orit is not water sensitive. For example it can be placed in plastic vialsor sacks, e.g. in thin, long hoses which also keep water away from theinitiator explosive and the blasting fuse. The material can be PVC orany appropriate plastic foil.

Depth of explosion and the area to be exploded are defined by thelocalization of the pollution (or the soil to be reclaimed). If desiredblasting can be carried out at arbitrary depth, i.e. not only surfacelayers of the soil but its deeper layers, expediently to the firstimpermeable layer, can be treated. With microorganism surviving underanoxic conditions remediation can be carried out in deep layers of thesoil, this way.

Thus, preferred explosives are those blasting materials which do nothave an expressly high explosion rate but which do not or only slightlydamage microorganisms. This effect, of course, depends also on thearrangement of holes and materials therein. The explosion rate of apreferred blasting material results in, besides destructive effect, asignificant if not dominant pushing effect (slow-action explosives).Thus, explosives used in mining are preferred. Explosion rate ispreferably less than 7000 m/s, preferably less than 6000 m/s, preferablyless than 5000 m/s, but at the same time larger than 500 or 1000 m/s,more preferably larger than 2000 or 3000 m/s, e.g. 3500 to 4000 m/s. Asa matter-of-course, effect and strength of the explosion, besidesexplosion rate, is a function of the said geometry and the quantity ofthe explosive, and is affected by other properties of the explosivese.g. explosion heat, specific gas volume, specific pressure etc.Selection of an explosive with appropriate parameters to the given taskand determination of the desired arrangement is routine for a skilledperson.

It is preferred if the material of the explosive, after explosion,results in compounds not detrimental to the soil and the microorganisms(not toxic), and preferably useful compounds are formed. Such explosivesare e.g. those comprising nitrate ion or group, e.g. NH₄NO₃ comprisingexplosives, e.g. paxit.

Taken together, a person skilled in pyrotechnics can easily decide onthe conditions and necessary strength of the blasting, on the basis ofthe present teaching.

If desired, as a next step we may apply after-treatment. This can be forexample aeration, infiltration, injection, or steaming.

Aeration is in order if for instance the microorganisms are aerobic, orthe conditions in the soil are such that oxygen is essential. Thefissures and cracks generated with detonation may not be sufficient toprovide the necessary oxygen. In this case oxygen is pumped into thesoil subsequently, for instance by placing a perforated tube in thesoil. The air is pumped in using a compressor.

This is especially important if no cracks arise in the soil or theydisappear quickly, for instance if the ground is loose, like sand ormoist soil. Subsequent aeration is also advisable if the pollution is inthe lower layers of the earth, and the detonation takes place there.

After the detonation it can be useful to administer active agentspreferably by infiltration or injection into the soil to improve theliving conditions of the microorganisms and/or to promote decomposition,such as by addition of dilute solutions of nitrate, sulphate, orphosphate. This can be important in cases where the soil has depletedits sources of these compounds or during detonation the compoundsintroduced into the ground aren't sufficient. Subsequent improvement ofthe soil is important in other aspects, we can add compounds that arefavored by the microorganisms.

If appropriate amount of fissures and cracks were generated in theground, infiltration can be a right choice for additional treatment.Otherwise injection is in order, which can be performed with aperforated tube.

The moisture and/or temperature of the soil can be improved by steaming.

The method of the invention can be used simply for the improvement ofthe ground. In this case the explosives along with the soil-improvingagents are placed in the ground and detonated as aforementioned.

The bioexplosion technology can be used for all biologicallydecomposable pollutants. And for this all microorganisms can be usedthat can decompose and/or inactivate pollutants effectively. Thisprocedure is versatile. Such microorganisms are well known, and manymore will be isolated in the years to come. The technology can be usedwith them, as well.

The microorganisms used for decomposition of the pollutants can beisolated from the environment, preferably from the polluted soil, or wecan use the commercially acquirable ones, or the genetically improved,previously mentioned strains (3, 4).

Of course it is advantageous if the microorganism used is resistant tothe pollutant (7), and if they are able to produce surfactants orenzymes capable of decomposition, or preferably both.

Considering their oxygen need we can distinguish aerobic, anaerobic, orfacultative anaerobic microorganisms (11). Whether we consider thetemperature tolerance of the microorganisms used, we can talk about theones that prefer cold (psychrophilic), the ones that prefer mediumtemperature (mesophilic), or the ones that prefer temperature abovenormal (thermophilic). In bioremediation mesophilic-aerobic,thermophilic-anaerobic, and facultative anaerobic microorganisms exceedthe others in effectiveness and rentability.

In certain cases there can be a demand that the microorganisms used forbioremediation be apathogenic (1, 2), in other words they shouldn'tcause neither plant, nor animal, nor human diseases. In other cases evenmicroorganisms capable of causing diseases can be used, if later on theydie or if they have no effect on humans, thus can be used as a pesticideor herbicide at the same time.

Microorganisms can be genetically enhanced, favorably carrying DNAfragment—of which the sequence is known—ligated into its' genomes as amarker.

To achieve effective bioremediation the life circumstances can beimproved: by setting temperature, moisture, oxygen concentration, or byadding additives like nutrition, macro and microelements (compoundscontaining carbon, nitrogen, phosphorous, sulphur, etc.). (11) (seebelow)

The activity of bioremediation of the microorganisms can be improvedwith tensides administered to the polluted water or contaminated soil incase of hydrophobic or badly soluble pollutants. (8, 9)

In anaerobic conditions (for instance lower layers of the earth or wetsoil) the activity of facultative anaerobic microorganisms can beinsured with electron acceptors and hydrogen acceptors—which allowanoxic respiration—such as nitrite (NO₂ ⁻), nitrate (NO₃ ⁻), phosphate(PO₄ ³⁻) or sulphate (SO₄ ²⁻) salts. (10)

Other additives promoting anoxic respiration, (NO₂, NO₃, PO₃, P0₄, P₂O₄,P₂O₇, ClO₂, ClO₃, ClO₄, BO₄, B₂O₇) even their inorganic salts or evenorgan be used.

A favorable solution would be to add electron acceptor additives whichcatalyse inorganic respiration such as metal ions and their salts,preferably Zn ions or Ti2+ions for instance in the form of TiCl₂ salt.

Choosing the additives we have to take the quality of the soil and itscomposition into consideration. For example the anions that contain Nand P are rare, while the ones that contain S (SO₄ ²⁻) and cations suchas K⁺ and Ca²⁺ aren't. It is also important to provide ions for thebacteria, which though rare to be found in the soil are vital for thecatalytic function of enzymes, for instance Mn-, Mo-, Ti- and Zn-ions.

Microorganisms can be exploded along with an organic C source (forexample: glucose, sacharose, molasses, acetate salts, glycerol).

Several of the additives, which improve the microorganisms' function anddecomposition activity—used during the bioexplosions—are summarizedaccording to function in Table 1 below. TABLE 1 Compounds promotinganoxic respiration: preferably redox systems and/or electron acceptorsand/or hydrogen acceptors, suitably compounds of metals with moreoxidative state (for example: Fe, Cu, Ti, Mn, or Mo - ions or manganatesand/or molybdenates), also nitrite-, nitrate-, chlorite-, chlorate-,perchlorate-, phosphate-, pyrophosphate-, sulphite-, sulphate-,pyrosulphate-ions or their salts. Metal ions, trace elements enhancingenzyme activity: suitably Fe—, Cu—, Ni—, Co—, Mn—, Mg—, Zn—, or Ca—ions, preferably Mn²⁺, Mg²⁺, Zn²⁺ and Ca²⁺. Carbon sources: preferablyglucose, saccharose, molasses, glycerol, acetate, xantane. Nitrogensources: suitably peptone, nitrite, nitrate, ammonium ions or theirsalts. Phosphorous sources: preferably phosphate, pyrophosphate ions ortheir salts. Sulphur sources: sulphate, pyrosulphate ions or theirsalts. Tensides and/or surfactants: mainly Tween 20, Tween 40, Tween 60,Tween 80, nonite, DMSO. Compounds promoting adhesion to surface:preferably all natural or synthetic polymers for instancepoly-acrilamide, poly-vinylpolymer, more preferably biologicallydecomposable polymers such as hydrocolloids, highly preferably xantane.

The additives in the concentrations that are used aren't toxic. Forexample the diluted solution of DMSO (dimethyl-sulphoxide) up to 20%isn't toxic.

Organic additives are environment friendly and decompose over time.

These additives can be partially added to the soil subsequently, afterthe detonation. The loose structure of the soil after the bioexplosionhelps the process.

Frequent pollutants purification with bioexplosion:

-   -   all paraffins asphaltenes, hydrocarbons containing maltene.    -   polyaromatic hydrocarbons, phenol, phenol derivatives    -   automobile, airplane fuels    -   halogenated hydrocarbons or their derivatives with sulphur        substitution (dichlorophenol, benzthiophene etc.)    -   organic molecules promoting the octane number        (methyl-tertiary-butylether etc)    -   dioxins    -   heterocyclic compounds (medicines, medicine components etc.)    -   pesticides, fungicides, herbicides    -   cyano compounds

Of course the technology can be used as a combination of other methods.

Another advantage of the technology is that not only the upper layers ofthe ground, but the lower layers can be treated, thus remediation can bedone in a way that the upper layers aren't touched.

-   -   (ii) If genetically non-modified microorganisms are isolated        from the environment for bioremediation, so called sterile        “solid minimal cultural-media” or preferably “silicagel solid        culture-media” is used. (for example in Petri-dishes)

Whether we isolate microorganisms capable of both aerobic and anoxicactivity it is advised to use culture-media containing nitrogen,sulphur, phosphorous salts and agar-agar, preferably sterile silicagelsolid culture-media.

It is important to administer the specific hydrophobic pollutant orother hydrophobic compounds (hydrocarbons, rock-oil, or its componentsand their derivatives) to decompose, dissolved in some kind of solvent,for instance a certain volatile organic solvent (alcohol, acetone,ether), preferably in pentane, hexane, or in methyl-benzene in the formof a thin film. Then the selected microorganisms from a fresh cultureshould be streaked onto this pollution layer, afterwards it should beincubated in the appropriate conditions for the strains (psychrophil,mesophil, thermophil, and aerobic, or anaerobic). After a certain timethe microorganisms resistant to the pollutant and are able to decomposeit will form colonies usually consistent or showing characteristicmorphology or pigmentation.

The microorganisms release enzymes into the area around the colonies,which are capable of decomposing the hydrophobic compounds such ashydrocarbons, and tensides are released, too. (FIG. 1. and 2.)

The enzyme production can be characterized by the width of the band(clearing up or discoloration) surrounding the colonies. Thischaracterizes the intensity of the enzyme production mainly (FIG. 1.).The produced enzyme activity can be determined by taking samples fromthe surrounding area of the colonies and we determine the composition ofthe pollutant by the means of gas chromatography. (FIG. 3 a and 3 b) Themicroorganisms showing the highest enzyme activity are then selected.

The microorganisms producing tensides can be selected according to thehydrophilic-hydrophobic examination. (for instance by water drops thenby paraffin drops; see FIG. 2).

Depending on the conditions of the selection of the microorganisms wecan acquire information concerning their essential conditions besidestheir activity of decomposition. Thus microorganisms used forbioremediation can be ones that prefer cold (psychrophilic), the onesthat prefer medium temperature (mesophilic), or the ones that prefertemperature above normal (thermophilic).

Considering their oxygen needs aerobic, anaerobic, and facultativeanaerobic microorganisms can be acquired. In bioremediationmesophilic-aerobic, thermophilic-anaerobic, and facultative anaerobicmicroorganisms should be used, because they exceed the others ineffectiveness and rentability. Facultative anaerobic microorganisms arepreferable. For their selection, besides the requirements postulated,(such as the culture-media should contain compounds promoting anoxicrespiration) it is also important that the colonies be incubated inanaerobic conditions.

In certain cases there can be a demand that the microorganisms used forbioremediation be apathogenic (1, 2), in other words they should causeneither plant, nor animal, nor human diseases. In other cases evenmicroorganisms capable of causing diseases can be used, if later on theydie or if they have no effect on humans, thus can be used as a pesticideor herbicide at the same time.

Using the above-mentioned selection method the inventors isolatedBacillus subtilis, Bacillus cereus, Pseudomonas sp. and Xanthomonas sp.microorganisms from oil polluted soils, the following of which weredeposited on Apr. 17, 2002 at the National Collection of Agriculturaland Industrial Microorganisms in Budapest according to the BudapestTreaty: MOL-number Deposition number MOL-2 NCAIM (P) B 1304 MOL-32 NCAIM(P) B 1305 MOL-51 NCAIM (P) B 1306 MOL-66 NCAIM (P) B 1307 MOL-107 NCAIM(P) B 1308

Microorganisms can be genetically modified, favorably carrying DNAfragment—of which the sequence is known—ligated into its' genomes as amarker.

During the selection of the facultative anaerobic microorganisms we canuse the following compounds, for instance electron acceptors andhydrogen acceptors, which allow anoxic respiration such as nitrite (NO₂⁻), nitrate (NO₃ ⁻), chlorite (ClO₂ ⁻), phosphate (PO₄ ³⁻) or sulphate(SO₄ ²⁻) etc. salts, furthermore inorganic salts of other compounds,which also help anoxic respiration (NO₂, NO₃, PO₃, PO₄, P₂O₄, P₂O₇,ClO₄, organic compounds can be used.

A favorable solution can be to add electron acceptor additives whichcatalyze inorganic respiration such as metal ions and their salts,preferably Zn²⁺ ions or Ti²⁺ ions for instance in the form of TiCl₂salt.

During the selection, while choosing the additives we have to take thequality of the environment and its composition into consideration inwhich we want to apply the microorganism.

If the environment to be treated is soil; for instance in thesubstratum, the anions that contain N and P are rare, while the onesthat contain S (SO₄ ²⁻) and cations such as K⁺ and Ca²⁺ aren't. It isalso important to provide ions for the bacteria, which though rare to befound in the soil are vital for the catalytic function of enzymes, forinstance Mn-, Mo-, Ti- and Zn-ions.

Several of the additives, which improve the activity of microorganismsisolated with our selection method for bioexplosions are summarizedTable 1 above.

It is evident that the additives should be added to the soil in aconcentration that isn't toxic to the microorganisms.

EXAMPLES Example 1

Cultures on Minimal Medium

Suspensions (1-20%) of soil samples containing pollutants (rock-oilcomponents, paraffins, asphaltenes, maltenes, etc, or derivatives of therock oil) dispersed in physiological salt solution or even in anyphysiologically useable buffer with a pH 6.5-7.6 were made. Certaindilutions of such suspensions were administered onto the surface ofagar-agar minimal culture-media, and were incubated at 0-80° C. forrandom time, preferably for 12-72 hours. The isolated colonies wereselected according to their activity of pollutant decomposition.

Agar-agar minimal culture-media (for 1000 g of distilled water):

-   -   0.1-3 g preferably 2.5 g Na₂HPO₄    -   0.1-3 g preferably 1.5 g KH₂PO₄    -   0.1-3 g preferably 0.5 g (NH₄)₂SO₄    -   0.01-3 g preferably 0.05 g CaCl₂    -   0.5-3 g preferably 2.0 g agar-agar    -   0.1-5 g preferably 1.5 g NaNO₃

It can be seen that the media contains ions promoting anoxic respiration(PO₄ ³⁻ and its protonated forms, SO₄ ²⁻, NO₃ ⁻) in other words itcontains electron acceptors, which also allows the selection of aerobicand facultative aerobic microorganisms.

In certain cases the aforementioned media was supplemented with 50 mLsometimes 10 mL of the following solution (1000 mL):

-   -   0.1-0.5 g preferably 0.25 g H₃BO₄    -   0.1-1.0 g preferably 0.25 g CoCl    -   0.1-2.0 g preferably 0.25 g CuCl₂    -   0.05-2.0 g preferably 0.25 g FeSO₄    -   0.01-1.0 g preferably 0.025 g MnCl₂    -   0.01-1.0 g preferably 0.025 g NaMoO₄    -   0.01-1.0 g preferably 0.025 g NiCl₂    -   0.01-1.0 g preferably 0.025 g TiCl₄

The metal ions of other oxidative states (for example Ti, Mn, Mo ions)also promote anoxic respiration as redox systems.

Example 2

Silicagel Culture-media

The microflora of the polluted soil samples can be grown on so called“silicagel minimal culture-media” which is a version of Vinogradszkijtype silicagel solid culture-media (12), which is supplemented with thecompounds mentioned in Example 1.

Thermophilic (50-80° C.) and extreme thermophilic (80-110° C.)microorganisms can be grown and selected on silicagel minimalculture-media.

Example 3

Examination of the Activity of Pollutant Decomposition

The ability of decomposition of the microorganisms isolated from minimalculture-media can also be examined on such solid media. In this case weadminister the hydrophobic pollutant (hydrocarbons, lipoids etc.),dissolved in some kind of solvent, for instance a certain volatileorganic solvent (alcohol, acetone, ether), preferably in pentane,hexane, in the form of a thin film. Then the microorganisms to beexamined should be streaked onto this pollution layer. (FIG. 1)

The colonies are incubated at the desired temperature with the givenoxygen concentration, for a desired time, preferably for 12-96 hours,more suitably for 48 hours, then the method should be repeatedpreferably 2-3 times again with the cultures grown.

The controlled level of oxygen concentration allows us to perform ourmethod in aerobic and anoxic conditions, thus we can isolatemicroorganisms which show activity in both aerobic and anoxicconditions. During the isolation of such facultative anaerobicmicroorganisms, part of the growth was done in anoxic conditions, andthe media contained compounds that promote anoxic respiration.

When the microorganisms isolated in the aforementioned way were streakedonto the film of pollutant, in the area around the colonies clearing upand discoloration could be observed showing that the pollutant waseither converted, or decomposed. (FIG. 1)

Below we will introduce how we examined the effectiveness ofdecomposition, the ratio of pollutants decomposed after a certain timein the clearing (FIG. 2), thus the selected enzymes' activity wasexamined. Also we could examine the appearance of other compounds,specifically tensides, during the course of decomposition, which helpedthe process. (FIG. 2)

Of course an expert can use other protocols in this case.

Example 4

Examination of the Effect of Microorganisms

The Activity of Enzymes of Oil Decomposition

On the surface of 15 mL of minimal agar-agar or minimal silicagelculture-media in a sterile Petri-dish with a 10 cm diameter weadministered a thin film of pollutant (rock oil products dissolved in 5%hexane or methyl-benzene solutions). Onto this film with a platinum loopwe streaked the microorganisms isolated from a polluted environment(soil, ground water, etc), and grown in liquid media. Then they wereincubated under the desired conditions (aerobic, or anaerobic), at thechosen temperature (15-20, 30-35 or 50-85° C.) for the desired time(24-240 hours), up until the microorganisms formed distinguishablecolonies. In case we can observe a certain change in the hydrocarbonfilm (clearing, discoloration) we take samples from these zones, thenextract it (hexane, methyl-benzene etc) with a solvent, then we examinethe rock oil product's quantity and its composition with the help of gaschromatography.

The effectiveness the production (also including the viability) ofenzymes capable of decomposing oil can be characterized by the width ofthe zone of clearing. The activity of the enzymes can be followed by thedecrease of the quantity of hydrocarbon components of the rock oilproducts.

The activity of a few of the isolated microorganism strains is comparedto other known strains (Table 2, 3). The letters in the table mean thefollowing: Ref I Hegrem* Ref II Hegboost* A MOL-2 NCAIM (P) B 1304 BMOL-32 NCAIM (P) B 1305 C MOL-51 NCAIM (P) B 1306 D MOL-66 NCAIM (P) B1307 E MOL-107 NCAIM (P) B 1308 F MOL-113 A Pseudomonas sp. strainisolated by the inventors*commercialized by Oil Cleaning Bio-Products Ltd. P.O.Box 46, Royston,Hertfordshire SG8 9PD U.K., see also the product descriptions and thehome page: www.ocbp.co.uk.

TABLE 2 The effect of bacteria groups on paraffins with differentmelting points. Sign of paraffin group DW 6266 DW 7580 DW 5456 DW 5658DW 5052 BO-1^(e) + + + + + 6 5 6 6-8 4-7 RO-1^(e) + + ++ + ++ 6 6  4-113-6  5-12 A^(t) ++++ + +++ ++++ +++ 15-18 5-8 10-15 11-19 11-16 B^(t)+++ + +++ ++++ +++  5-11 5-6 10-15 13-20 10-16 C^(t) +++ ± +++ +++ +++10-15 4 14-17 14-18 11-14 D^(e(t)) + + ++++ ++++ + 5-7 4-5 10-22 10-344-7 E^(e(t)) + +++ +++ +++ +++ 6-7 10-13 13-17 11-13 13-16 F^(e(t)) ++++ ++ ++ ++  9-12  6-10  7-12  4-10  7-12^(t)effect of tensides^(e)enzyme activityactivity+ insignificant++ partial+++ satisfactory++++ outstandingnumber - the diameter of the decomposed area

TABLE 3 Different precipitated rock oil decomposition with bacteriagroups at 37° C. after 96 hours. 5% asphaltene + Alg Signhydrophobic^(x) asphaltene maltene #571 oil BO-1 + + ++ 4-7 9 4-7  6-12RO-1 + + ++++  4-10 7 4-8 15-18 A + ++++ ± ++++ 5 10-38 2 22-25 B +++++ + ++++ 4-8 14-20 4-7 34-37 C + ++ ± ++++ 4-6  7-12 2-4 25-30D + + + ++++ 3-6 4 4-8 30-35 E ++++ + ++++ ++++ 22-25 5-7 20-25 30-35F + + ++++ 4-5 4-5 10-35 20-35t-effect of tensidese—enzyme activityactivity+ insignificant++ partial+++ satisfactory++++ outstandingnumber - the diameter of the decomposed area

While comparing the FIG. 3 with the Tables one can see that the enzymeactivity (measured with gas chromatography, i.e. GC) of our isolatedstrains was a match for the strains known up to date, the effectivenessof decomposition (characterized by the average width of the area clearedup as a band), considering the pollutant significantly exceeded that ofthe strains known up to date. In case of the Hegrem and Hegboostproducts we weren't able to detect any production of tensides. With ourtechnology microorganisms specifically selected can be produced and canbe used specifically for a pollutant that they decompose the mosteffectively.

The Detection of Tensides Produced with the Help ofHydrophilic-hydrophobic Drops

We repeat the procedure mentioned in Example 4 in case of the oildecomposing enzymes, with the exception that in the clearing surroundingthe colonies of the chosen microorganisms, under the desired conditionswe administer a few drops of distilled water or melted paraffin onto thesurface of the media. In the zone containing tensides the drop ofdistilled water spreads out, while in the area with no clearing up(hydrophobic) it forms a moveable bead like drop. The melted paraffindrop spreads out in a moveable manner in the area with tensides, whileit sticks to the hydrophobic zone making its movement impossible. (FIG.2)

The surface critical angle of the drops is measurable, and can even beused to quantitively describe the production of tensides if fixing otherparameters. (growth time, drop zone).

Example 5

Method of Bioremediation

We administer a mixture of microorganisms of B. cereus, B. subtilis, andPseudonomas sp. group isolated as mentioned in Examples 1-4 in anequivalent ratio and in satisfactory amount, preferably in aconcentration of 10⁴-10¹², suitably 10¹¹ microorganisms per kg ofcontaminated soil, to the polluted medium in a way that after desireddistances (0.5-6 m, preferably 1.5-2 m) and to a given depth (to thelevel of ground water) we drill holes. The holes are drilled in thecorners of a square, into one of these holes we place themicroorganisms, then we place the additives and nutrition into anotherhole, and in the rest of the holes we place the explosives (FIG. 4) in asymmetrical manner. Then follows the bioexplosion (FIG. 5 b)

After the bioexplosion we leave the soil undisturbed for 1-120 days,preferably 5-6 days, then comes the subsequent treatment (aeration,infiltration, injection, steaming, etc.).

The decomposition of a rock-oil pollution of 40-60 g/kg concentration at4-6 m underground after a bioexplosion without subsequent treatment canbe seen in FIG. 5 a.

The same pollution was produced (ex situ) and mixed together (with aprism) with the soil, and was cleansed with the same quality andquantity of microorganisms and additives used in the in situbioexplosion.

The specific cost of the two methods according to our calculations:

-   -   Ex situ (prism) method: 40-60 USD/tons    -   In situ (bioexplosion) method: 8-16 USD/tons

According to our experiences the bioexplosion technology's costs arecompetitive with other prism-remediation technologies, while theeffectiveness exceeds them.

Execution of the Detonation

We first survey the location, then the horizontal and vertical extent ofthe pollution of the soil. Afterwards holes are drilled into the groundin the desired distance, diameter, depth, in a quadratic manner, all theway down to the ground water level. We place the explosives of thedesired quality (such as paxite, etc) and in the desired quantity into aplastic tube, which is lowered into every uneven numbered hole. Thus wecan achieve the desired detonation effect. (FIG. 4)

The explosives are connected to an electric fuse, and the wires areconnected in parallel so the detonation can be simultaneous or indesired portions. (FIG. 3 b)

In the following the desired amount of additives are dissolved in water,then placed in plastic tubes, which are then placed in the even holes,either above the explosives or in empty ones. The microorganisms alsoplaced in plastic tubes, should also be placed in the empty evennumbered holes.

At last the additives and microorganisms can be bioexploded, thestrength of the detonation should be chosen to achieve maximum mixing ofthe microorganisms and additives without allowing them to the surface.

Example 6

Procedure of Aeration

Down to the level of the ground water we place metal tubes in aquadratic formation into the polluted ground loosened by the explosion.At a selected part of the tubes (third, or fourth of the length) wedrill a desired number of holes with the right diameters. With the helpof a compressor we can administer oxygen, water vapor, or with a pump wecan administer new portions of additives and/or compounds promoting thedecomposition of the pollutant.

Example 7

Application

The technology can be used to cleanse polluted soil, ground water, trashdumps of rock oil, grease, fuels, other hydrocarbons, and derivatives(halogenated), or of pesticides, herbicides, toxic wastes, or of usuallybiologically decomposable/neutralizeable xenobiotics. The use of thistechnology can be confined within limits. In populated areas or gasstations the use of the technology is prohibited or limited.

In addition our technology can be used to moderate the effect ofenvironmental catastrophes causing ground contamination (outburst ofnatural gas and thermal water etc.), the effect of serious soilpollutions (such as pipeline deficiency, cyan pollution etc.), or theeffect of polluted floods, inland waters, waste-piles etc, and to try tocleanse the ones that are situated between the surface and the groundwater level. In certain cases it can also be used against pollutants,which have already reached the ground water.

Cited Literature

-   1.) Oil Cleaning Bio-products Ltd., Press release on Hegrem bacteria-   2.) Oil Cleaning Bio-products Ltd., Press release on Hegboost    bacteria-   3.) Thomas J. M. and Ward C. H. (1989), Environ. Sci. Technol.    23:760-766.-   4.) Van der Meer et al. (1992), Microbiol. Rev. 56:677-694.-   5.) Kopp-Holtwiesche B. et al. (1992), Biotech. Forum Europe No. 6.-   6.) Sloan R. (1987), Oil and Gas J. 61-66.-   7.) Bouwer E. J. and Zehnder A, J. B. (1993) TIBTECH 11:360-367.-   8.) Plumb R. H. J. (1991), Groundwater monitoring Rev. 11:157-164.-   9.) Rijnarts H. H. et al. (1990), Environ.Sci.Technol. 24:1349-1354.-   10.) Bouwer E. J. and Ward C. H. (1989), Environ Sci. Technol.    23:760-766-   11.) Bergey's Manual of Systematic Bacteriology Vol. 1 (1984)-   12.) A. S. Dietz, A. A. Yayanos, Appl. Environm. Microbiol. 36:966    (1978)

1. A process for decreasing the degree of the pollution comprising thesteps of (i) placing in the polluted soil or close to it, below thesurface of the soil microorganisms useful for decomposing orinactivating at least one kind of material causing pollution, saidmicroorganisms being, if desired, resistant to said material, and anexplosive (ii) mixing the polluted soil and the microorganisms byexplosion, whereby the soil treated is loosened and (iii) allowing themicroorganisms to act in the soil.
 2. The process of claim 1 wherein instep (i) also additive materials improving living conditions ofmicroorganisms and/or facilitating their propagation and/orsoil-improving function are placed below the surface of the soil, and instep (ii) said additive materials are also mixed with the microorganismsand the polluted soil.
 3. The process according to claim 1 or 2 whereinthe microorganisms, explosive and optionally further additive materialsare placed in holes bored in soil.
 4. The process according to claim 3wherein the microorganisms and the explosive are placed in separateholes.
 5. The process according to claim 3 wherein the microorganismsand the explosive are placed in the same holes above each other,preferably the explosive being placed below the microorganisms.
 6. Theprocess according to any of claims 3 to 5 wherein the microorganisms,the explosive and preferably the additive materials are placed inplastic tubes which are then placed in the holes.
 7. The processaccording to any of claims 2 to 6 wherein after the explosion additivesimproving living conditions of the microorganisms and/or facilitatingdecomposition are introduced into the soil, for instance by aeration,infiltration or injection, wherein preferably one or more of thefollowing are applied: compounds promoting anoxic respiration, metalions, trace elements, carbon sources, nitrogen sources, phosphoroussources, sulphur sources tensides and/or surfactants, compoundspromoting adhesion to surface.
 8. The process according to any of claims3 to 6 wherein the holes are at a distance of 0.5 to 5 m from each otherand the strength of explosion is set to a value which results in thedamage of at most a small part of microorganism, more preferably in nodamage and no uncovering of the microorganisms.
 9. The process accordingto any of claims 1 to 8 wherein the pollutant is a mineral oil componentor derivative.
 10. A method for preparing a facultative anaerobicmicroorganism in an isolated form, said microorganism being useful fordecomposing a hydrophobic pollutant comprising a mineral oil componentor derivative thereof during soil remediation capable of exerting itsdecomposing activity at the boundary of the hydrophobic phase comprisingthe said hydrophobic pollutant, mineral oil component or derivative anda hvdrophilic phase, the process comprising the steps of i) applying afilm comprising said hydrophobic pollutant to a minimal medium lackingcarbon source, ii) inoculating this medium with a sample comprising amixture of microorganisms said sample being obtained from an oilpollution, and incubating the medium after inoculation at least untildetectable microorganism colonies are formed, if the formation ofcolonies does not occur within an arbitrarily defined time period stepi) and present step ii) are repeated, iii) decomposing activities of themicroorganism from the colonies formed are tested at the surround of thecolonies and iv) tenside producing abilities of the decomposingmicroorganisms obtained from the colonies are checked and a tensideproducing microorganism is selected, wherein the minimal mediumcomprises materials facilitating anoxic respiration, preferably electronacceptors and/or oxygen sources—in particular one or more of thefollowing: Ti-compounds, Mn-compounds, nitrite, nitrate, phosphate,pyrophosphate ions or their salts, and wherein the incubation is carriedout at least partly under anaerobic conditions.
 11. The method accordingto claim 9 or 10 wherein the tenside producing ability of themicroorganisms from the colonies obtained is be studied by ahydrophobic-hydrophilic drop test.
 12. The method according to claim 9or 10, wherein the decomposing activity is assessed by assaying thepollutant concentration of samples taken from the close surround orimmediate vicinity of the colonies and/or on the basis of the diameterof the decomposed area.
 13. The method of claim 12 wherein as adecomposing activity paraffin decomposing activity is assayed or anenzyme activity for decomposing typical mineral oil pollutions,preferably by sampling, solvent extraction and then by gaschromatography.
 14. Use of a facultative anaerobic microorganism of thePseudomonas genus for decomposing a hydrophobic soil pollutantcomprising mineral oil component or derivative during soil remediation,said microorganism producing at least one enzyme capable of decomposingthe mineral oil component or derivative, and at least one tenside. 15.Use of a facultative anaerobic microorganism of the Pseudomonas genus,said microorganism producing at least one enzyme capable of decomposingthe mineral oil component or derivative, and at least one tenside, fordecomposing a hydrophobic soil pollutant comprising mineral oilcomponent or derivative during soil remediation by any of the methodsaccording to claims 1 to
 9. 16. The use of any of claims 14 to 15wherein the oil-pollutant decomposing activity of the microorganism,detected by culturing on a polluted medium on any of the followingoil-pollutants: hydrophobic deposit, asphaltene, maltene, 5% asphalteneplus oil, is at least 1.5 times larger, as an average, than that of theHegrem or Hegboost microorganism.
 17. The use of any of claims 14 to 17wherein the microorganism is any of the following microorganismsdeposited on Apr. 17, 2002 at the NCAIM: NCAIM (P) B 1304, NCAIM (P) B1305, NCAIM (P) B 1306, NCAIM (P) B 1307, NCAIM (P) B 1308, or anystrain derived therefrom.
 18. A facultative anaerobic microorganism ofthe Pseudomonas genus obtainable by the method of any of claims 10 to13, said microorganism producing at least one enzyme capable ofdecomposing the mineral oil component or derivative and at least onetenside, being capable of decomposing a mineral oil component orderivative during soil remediation.
 19. The microorganism of claim 18which is any of the following microorganisms deposited on Apr. 17, 2002at the NCAIM: NCAIM (P) B 1304, NCAIM (P) B 1305, NCAIM (P) B 1306,NCAIM (P) B 1307, NCAIM (P) B 1308, or any strain derived therefrom. 20.The microorganism of any of claims 18 to 19 which is geneticallymodified preferably carries, incorporated into its genome, a DNAfragment of a known sequence.
 21. Kit for decomposing a hydrophobic soilpollutant comprising mineral oil component or derivative during soilremediation comprising an information carrier with users instructionswhich comprise instructions for carrying out any of the steps of any ofthe processes of claims 1 to 9 and said kit further comprising at leastone kind of material applicable in any of the processes of claims 1 to9.
 22. The kit of claim 21 comprising a microorganism as defined in anyof claims 14 to
 20. 23. The kit of claim 22 comprising on or more of thefollowing group: explosives, aids to blasting, additives to ensureliving conditions or helping microorganisms or increasing the effectthereof.
 24. The kit of claim 23 comprising one or more of the followingcompounds promoting anoxic respiration, metal ions, trace elements,carbon sources, nitrogen sources, phosphorous sources, sulphur sourcestensides and/or surfactants, compounds promoting adhesion to surface.25. The process according to claims 8 wherein the holes are at adistance of 1.5 to 2 m from each other.